<i>N</i>‑Heterocyclic Carbene Catalyzed Intramolecular Acylation of Allylic Electrophiles

The <i>N</i>-heterocyclic carbene (NHC) catalyzed addition reaction has been well documented recently; however, the NHC-catalyzed substitution reaction especially the S_N2′ type reaction remains a challenge. As one of the most fundamental reaction types in organic chemistry, the S_N2′ reaction catalyzed by NHC would be a powerful tool in organic synthesis. Therefore, the first NHC-catalyzed intramolecular S_N2′ substitution reaction of aldehyde with allylic electrophiles has been developed. A variety of α,β-unsaturated chromanones were obtained under a domino S_N2′ reaction and isomerization. Mechanistic experiments were conducted to confirm the nature of this S_N2′ reaction

Catalytic Asymmetric [4 + 1] Annulation of Sulfur Ylides with Copper–Allenylidene Intermediates

The first copper-catalyzed asymmetric decarboxylative [4 + 1] cycloaddition of propargylic carbamates and sulfur ylides was successfully developed. This strategy led to a series of chiral indolines with synthetically flexible alkyne groups in good yields and with high enantio- and diastereoselectivities (up to 99% yield, 98% ee, and >95:5 dr). A possible mechanism and stereoinduction mode with copper–allenylidenes were proposed as the possible dipolar intermediate

Catalytic Asymmetric [4 + 1] Annulation of Sulfur Ylides with Copper–Allenylidene Intermediates

The first copper-catalyzed asymmetric decarboxylative [4 + 1] cycloaddition of propargylic carbamates and sulfur ylides was successfully developed. This strategy led to a series of chiral indolines with synthetically flexible alkyne groups in good yields and with high enantio- and diastereoselectivities (up to 99% yield, 98% ee, and >95:5 dr). A possible mechanism and stereoinduction mode with copper–allenylidenes were proposed as the possible dipolar intermediate

Sequential Visible-Light Photoactivation and Palladium Catalysis Enabling Enantioselective [4+2] Cycloadditions

Catalytic asymmetric cycloadditions of reactive ketene intermediates provide new opportunities for the production of chiral heterocyclic molecules. Though known for over 100 years, ketenes still remain underexplored in the field of transition-metal (TM)-catalyzed asymmetric cycloadditions because (1) ketenes, as highly electron-deficient species, are possibly unstable to low-valence TMs (i.e., decarbonylation or aggregation) and (2) the conventional thermal synthesis of ketenes from acyl chlorides and amines may be incompatible with TM catalysis (i.e., reactive acyl chloride and amine hydrochloride byproducts). Herein, we detail the unprecedented asymmetric [4+2] cycloaddition of vinyl benzoxazinanones with a variety of ketene intermediates via sequential visible-light photoactivation and palladium catalysis. It is well demonstrated that the traceless and transient generation of ketenes from α-diazoketones through visible-light-induced Wolff rearrangement is important for the success of present cycloaddition. Furthermore, chiral palladium catalysts with a new, chiral hybrid P, S ligand enable asymmetric cycloaddition with high reaction selectivity and enantiocontrol

Sequential Visible-Light Photoactivation and Palladium Catalysis Enabling Enantioselective [4+2] Cycloadditions

Catalytic asymmetric cycloadditions of reactive ketene intermediates provide new opportunities for the production of chiral heterocyclic molecules. Though known for over 100 years, ketenes still remain underexplored in the field of transition-metal (TM)-catalyzed asymmetric cycloadditions because (1) ketenes, as highly electron-deficient species, are possibly unstable to low-valence TMs (i.e., decarbonylation or aggregation) and (2) the conventional thermal synthesis of ketenes from acyl chlorides and amines may be incompatible with TM catalysis (i.e., reactive acyl chloride and amine hydrochloride byproducts). Herein, we detail the unprecedented asymmetric [4+2] cycloaddition of vinyl benzoxazinanones with a variety of ketene intermediates via sequential visible-light photoactivation and palladium catalysis. It is well demonstrated that the traceless and transient generation of ketenes from α-diazoketones through visible-light-induced Wolff rearrangement is important for the success of present cycloaddition. Furthermore, chiral palladium catalysts with a new, chiral hybrid P, S ligand enable asymmetric cycloaddition with high reaction selectivity and enantiocontrol

GEN1 depletion results in supernumerary centrosomes in late G2 or mitosis. A.

<p>HeLa cells were treated with GEN1 or control (CTRL) siRNA for 48 h, methanol fixed and immunostained with the indicated antibodies. Identification of centrosomes and centrioles was based on immunostaining with γ-tubulin and Centrin 2 antibodies, respectively. Scale bar, 10 µm. <b>B.</b> Quantification of centrosomes with 1, 2 or more than 2 centrioles in CTRL or GEN1 depleted interphase cells. Graph represents the mean of three independent experiments. (n = 300 centrosomes/condition/experiment). Error bars indicate S.E.M. <b>C.</b> Quantification of interphase and mitotic cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049687#pone-0049687-g003" target="_blank"><b>Fig. 3A</b></a>) with extra centrosomes (interphase>1 centrosome (G1) or>1 linked centrosome pair (S-G2), mitosis>2 separated centrosomes). Graph represents the mean of three independent experiments (n = 300 cells/condition/experiment). Error bars indicate S.E.M. <b>D.</b> The percent of supernumerary centrosomes with 1, 2 or more than 2 centrioles after GEN1 depletion. Graph represents the mean of three independent experiments. (n = 300 centrosomes/condition/experiment). Error bars indicate S.E.M. <b>E.</b> Quantification of mitotic cells with>2 mitotic spindles 48 h after treatment with CTRL, GEN1 and/or ATR siRNA followed by incubation with or without ATM inhibitor(10 uM Ku55933. Mitotic spindles were visualized by GEN1 (anti-GEN1 (651–892aa) antibody) and γ-tubulin immunostaining. Graph represents the mean of three independent experiments. (n = 300 cells/condition/experiment). Error bars indicate S.E.M.</p

A Novel Role of Human Holliday Junction Resolvase GEN1 in the Maintenance of Centrosome Integrity

<div><p>The maintenance of genomic stability requires accurate genome replication, repair of DNA damage, and the precise segregation of chromosomes in mitosis. GEN1 possesses Holliday junction resolvase activity <em>in vitro</em> and presumably functions in homology driven repair of DNA double strand breaks. However, little is currently known about the cellular functions of human GEN1. In the present study we demonstrate that GEN1 is a novel centrosome associated protein and we characterize the various phenotypes associated with GEN1 deficiency. We identify an N-terminal centrosome localization signal in GEN1, which is required and sufficient for centrosome localization. We report that GEN1 depletion results in aberrant centrosome numbers associated with the formation of multiple spindle poles in mitosis, an increased number of cells with multi-nuclei, increased apoptosis and an elevated level of spontaneous DNA damage. We find homologous recombination severely impaired in GEN1 deficient cells, suggesting that GEN1 functions as a Holliday junction resolvase <em>in vivo</em> as well as <em>in vitro</em>. Complementation of GEN1 depleted cells with various GEN1 constructs revealed that centrosome association but not catalytic activity of GEN1 is required for preventing centrosome hyper-amplification, formation of multiple mitotic spindles, and multi-nucleation. Our findings provide novel insight into the biological functions of GEN1 by uncovering an important role of GEN1 in the regulation of centrosome integrity.</p> </div

GEN1 depletion interferes with cell cycle progression and results in multi-nucleation and apoptosis. A.

<p>(left) 293T cells were treated with CTRL or GEN1 siRNA for 48 h stained with propidium iodine (PI) and analyzed by flow cytometry. (middle) G2/M cells were identified using Modfit software. (right) Mitotic cells were detected by phospho H3 antibody staining followed by flow cytometric analysis. Quantification is based on three independent experiments. Error bars indicate S.E.M. <b>B.</b> Hela cells were treated with CTRL or GEN1 siRNA for 48 h, labeled with or without 10 uM BrdU for 2 hours, stained with FITC conjugated anti-BrdU antibodies and propidium iodine (PI), and analyzed by flow cytometry. x-axis: propidium iodine (PI). y-axis FITC. <b>C.</b> CTRL or GEN1 siRNA treated Hela cells were incubated with 10 uM BrdU for 2 hours, and return to BrdU-free medium for the indicated times. Cell cycle progression of BrdU labeled cells were analyzed by flow cytometry. <b>D.</b> HeLa cells treated with CTRL or GEN1 siRNA (48 h) were methanol fixed and immunostained with GEN1(anti-GEN1 (651–892aa) antibody) and α-tubulin. DNA was visualized by DAPI. (Right) Graph represents the mean of three independent experiments. (n = 100 cells/condition/experiment). Error bars indicate S.E.M. <b>E.</b> HeLa cells were treated with CTRL or GEN1 siRNA for 48 h fixed, stained with annexin V and analyzed by flow cytometry. Quantification was based on three independent experiments. Error bars indicate S.E.M.</p

Human GEN1 localizes on the centrosome. A.

<p>(top) MRC5 cells were fixed with methanol and immunostained with GEN1 antibody (GST-GEN1 651–892 antibody) and DAPI. Scale bars, 10 µm. (bottom) Cells were lysed and separated by SDS-PAGE and immunoblotted using the indicated antibodies. <b>B.</b> HeLa cells were fixed with methanol and immunostained with anti-GEN1 (651–892aa) antibody, anti-γ-tubulin antibody and DAPI. Scale bars, 10 µm. <b>C.</b> Hela cells at different phases of the cell cycle were fixed with methanol and co-immunostained with the indicated antibodies and DAPI. Scale bars, 10 µm. <b>D.</b> Centrosomes were isolated from HeLa cells and purified fractions from a discontinuous sucrose gradient were separated by SDS-PAGE and immunoblotted using the indicated antibodies. <b>E.</b> Schematic showing GEN1 domains and alignment of the Cyclin E CLS and the putative CLS in GEN1. Highly conserved amino acids are shown in red. In black are shown three of the key amino acids within the endonuclease domain of GEN1. XPG (-N: N-terminal, -I, internal) nuclease domain domain very similar to the nuclease domain first identified in XPG. <b>F.</b> HeLa cells were transfected with the indicated plasmids and 24 h later methanol fixed and immonostained with γ-tubulin and DAPI. GEN1 was detected by GFP fluorescence. Scale bars, 10 µm. Images to the left are zoomed 27.5×. CLS-4A: centrosome localization defective mutant, Ci-3A: catalytic inactive mutant.</p

The role of GEN1 in maintaining centrosome integrity appears separable from its HJ resolvase function. A.

<p>HeLa cells were simultaneously treated with GEN1 siRNA and the indicated siRNA insensitive GFP-GEN1 constructs for 48 h, methanol fixed and immunostained with the γ-tubulin and DAPI. Quantification was based on three independent experiments. (n = 100 cells/condition/experiment). The white box shows a 6.8 magnification of the centrosome identified by γ-tubulin. Error bars indicate S.E.M. Scale bars, 10 µm. <b>B.</b> HeLa cells were treated as in A. but immunostained with α-tubulin and DAPI. Quantification was based on three independent experiments. Error bars indicate S.E.M. (n = 100 cells/condition/experiment). Scale bars, 10 µm. <b>C.</b> HeLa cells were treated as in A. but immunostained with γ-H2AX and DAPI. Quantification was based on three independent experiments. Error bars indicate S.E.M. (n = 100 cells/condition/experiment). Scale bars, 10 µm.</p